Rozman Grinberg Inna, Bimaï Ornella, Shahid Saher, Philipp Lena, Martínez-Carranza Markel, Banerjee Ipsita, Lundin Daniel, Stenmark Pål, Sjöberg Britt-Marie, Logan Derek T
Department of Biochemistry and Biophysics, Stockholm University, Sweden.
Biochemistry & Structural Biology, Centre for Molecular Protein Science, Department of Chemistry, Lund University, Sweden.
FEBS J. 2025 Mar 3. doi: 10.1111/febs.70037.
NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.
NrdR是一种细菌转录阻遏物，由一个锌（Zn）带结构域和一个ATP锥结构域组成。了解其作用机制有助于新型抗菌药物的设计。NrdR特异性结合核糖核苷酸还原酶操纵子上游的两个“NrdR框”，大肠杆菌有三个这样的操纵子：nrdHIEF、nrdDG和nrdAB，我们在最后一个操纵子中鉴定出一个新的框。我们发现，当加载ATP加脱氧三磷酸腺苷（dATP）或等效二磷酸组合时，大肠杆菌NrdR（EcoNrdR）与所有三个位点的结合强度相似。腺嘌呤核苷酸的其他组合均不能促进与DNA的结合。我们展示了EcoNrdR-ATP-dATP和EcoNrdR-ADP-dATP的晶体结构，这是NrdR的首批高分辨率晶体结构。我们还确定了与DNA结合的EcoNrdR-ATP-dATP和新型EcoNrdR-ATP细丝的冷冻电子显微镜结构。EcoNrdR的四聚体形式涉及锌带结构域对和ATP锥之间的交替相互作用。这些结构揭示了ATP锥与锌带结构域相对取向的相当大的灵活性。与DNA结合的EcoNrdR-ATP-dATP的结构表明，ATP锥和锌带之间的显著构象重排伴随着DNA结合，而ATP锥保持相同的相对取向。相比之下，加载ATP的EcoNrdR细丝显示出ATP锥对的重排，并隔离了NrdR的DNA结合残基，使其无法与DNA结合。我们的结果与之前的结构和生化研究相结合，表明EcoNrdR结构具有高度灵活性，当加载正确的核苷酸时，会适应最佳的启动子结合构象。
